The present invention relates to a method of improving the binding of a series of consecutive nucleotide bases to a complementary nucleic acid molecule, especially for use in improving the binding of capture oligonucleotides, in particular in methods for isolating primer extension products such as sequencing products, modular oligonucleotides and kits for performing methods of the invention.
The binding of complementary nucleotide bases to one another represents one of the most significant and fundamental findings in science this century and heralded the rapid development of the field of biochemistry. Whilst allowing an understanding of the mechanisms underlying the continuation of life, the discovery has also provided the basis for the development of valuable molecular biological tools.
The isolation and sequencing of naturally occurring nucleic acid molecules is a common goal for molecular biologists. The use of complementary oligonucleotides to isolate nucleic acid molecules is commonplace. Similarly, complementary oligonucleotides are frequently used to bind single-stranded nucleic acid molecules and act as primers for extension reactions to produce complementary strands to the template and forms the basis of such experimental procedures as polymerase chain reaction (PCR) and sequencing reactions.
However, the specificity of binding of oligonucleotides to template or target DNA depends on a number of parameters any one of which may result in poor efficiency of binding and consequently poor experimental results. The specificity of the interaction may conveniently be determined by the assessment of Tm, the temperature at which duplexes dissociate. This is however also dependent on other parameters, for example the buffer in which the reaction is performed. For a particular experimental system, Tm will be affected by various factors including the extent of complementarity, the sequence of the target and/or oligonucleotide, derivatization of the oligonucleotide and length of the oligonucleotide. The binding of oligonucleotides may therefore be improved, as evidenced by an increased Tm under the same experimental conditions, by altering these parameters. However, the variation which may be achieved by altering these parameters is limited. There therefore exists a need for further methods which will improve the binding of oligonucleotides to target DNA.
Surprisingly, is has now been found that modular probes or primers composed of at least two modules (oligonucleotides) which bind to adjacent regions of target DNA exhibit improved binding relative to a single oligonucleotide spanning the same length as the separate modules (see WO98/13522). For example, it has been found that two adjacent 18-mer oligonucleotides bind more efficiently to target DNA than the composite 36-mer oligonucleotide.
The use of primers composed of adjacent modules for sequencing purposes has been described previously (Kotler et al., 1993, Proc. Natl. Acad. Sci. USA, 90, p4241-4245; Kieleczawa et al., 1992, Science, 258, p1787-1791 and Szybalski, 1990, Gene, 90, p177-178). However, in these cases the modular primers were used to replace longer primers such that libraries of all sequences of the shorter primers could realistically be pre-synthesized as they had fewer possible sequence permutations than longer primers. In all cases, the modular primers were only shown to have, in sequencing reactions under the same conditions, efficacy as good as the longer primers. In contrast, in the present invention, surprisingly, even better binding is achieved, when a single oligonucleotide is split into separate components. Furthermore, the previous work indicates that the effect of modular primers may only be achieved if the modules do not have a single (or more) base(s) between them when bound to the template. For improved binding as described herein, so such restriction is applicable although even better binding is observed when no gaps exist between the modules.